Comamonas Terrae Sp. Nov., an Arsenite-Oxidizing Bacterium Isolated from Agricultural Soil in Thailand

J. Gen. Appl. Microbiol., 58, 245‒251 (2012)

Short Communication

Comamonas terrae sp. nov., an arsenite-oxidizing bacterium isolated from agricultural soil in Thailand

Kitja Chitpirom,1 Somboon Tanasupawat,2,* Ancharida Akaracharanya,3 Natchanun Leepepatpiboon,4 Alexander Prange,5 Kyoung-Woong Kim,6 Keun Chul Lee,7 and Jung-Sook Lee7

1 Inter-department of Environmental Science, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand 2 Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand 3 Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand 4 Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand 5 Microbiology and Food Hygiene, Niederrhein University of Applied Sciences, Rheydter Strasse 277, D-41065 Möenchengladbach, Germany 6 Department of Environmental Science and Engineering, Gwangju Institute of Science and Technology, Gwangju 500‒712, Korea 7 Korean Collection for Type Cultures, Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong, Daejeon 305‒806, Republic of Korea

(Received August 11, 2011; Accepted February 14, 2012)

Key Words—arsenite-oxidizing bacterium; Comamonas terrae sp. nov.; β-Proteobacteria; 16S rRNA gene

The genus Comamonas belonging to the family Co- 1987; Wauters et al., 2003; Young et al., 2008; Yu et al., mamonadaceae of the class Beta-Proteobacteria was 2011). Comamonas strains have been isolated from a proposed by De Vos et al. (1985) after a polyphasic phenol-activated-sludge process and soils contami- study. In the genus Comamonas, 13 species have nated with heavy metals (Kanazawa and Mori, 1996; been described at the time of writing, C. aquatica, C. Watanabe et al., 1999). During the course of screening badia, C. denitrifi cans, C. kerstersii, C. koreensis, C. of a high arsenic resistant bacteria from arsenic con- nitrativorans, C. odontotermitis, C. terrigena (type spe- taminated soils in Thailand, Comamonas strain A3-3T cies), C. testosteroni, C. composti, C. zonglianii, C. was isolated and reported (Chitpirom et al., 2009). This thiooxidans, and C. granuli (Chang et al., 2002; Chou paper, we propose Comamonas terrae sp. nov. to et al., 2007; De Vos et al.,1985; Etchebehere et al., strain A3-3T. 2001; Gumaelius et al., 2001; Kim et al., 2008; Narayan Strain A3-3T, an arsenite oxidizing bacterium was et al., 2010; Tago and Yokota, 2004; Tamaoka et al., isolated from the arsenic contaminated soil samples (< 5 mg/kg) in central of Thailand by an enrichment culture method using 0.1 g/L sodium arsenite (Chit- * Address reprint requests to: Dr. Somboon Tanasupawat, pirom et al., 2009). The MICs for arsenite and arsenate Department of Biochemistry and Microbiology, Faculty of Phar- of the A3-3T isolate were 40 and 400 mM, respectively maceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand. (Chitpirom et al., 2009). The capability to oxidize ar- T E-mail: [email protected] senite (+III) to arsenate (+V) of A3-3 was tested in The GenBank/EMBL/DDBJ accession number for the 16S situ by X-ray absorption near edge structure (XANES) rRNA gene sequence of strain A3-3T is GQ497244. analysis. The strain was phenotypically characterized 246 CHITPIROM et al. Vol. 58 by Gram staining, fl agella staining, cell morphology, and 50°C) were investigated in TYEG broth. Antibiotic colony appearance, spore formation and pigmenta- susceptibility was determined by disc diffusion assay tion on TYEG (tryptone-yeast extract-glucose) agar (Bauer et al., 1966). The antibiotic discs contained am- medium at 30°C for 24 h (Chitpirom et al., 2009). Cata- picillin (10 μg), bacitracin (10 U), carbenicillin (100 μg), lase, oxidase, hydrolysis of casein, starch, Tween 80, cephalothin (30 μg), clindamycin (2 μg), erythromycin nitrate reduction and deoxyribonuclease (DNase) ac- (15 μg), gentamicin (10 μg), imipenem (10 μg), kana- tivity were determined as described by Barrow and mycin (30 μg), novobiocin (5 μg), penicillin (20 U), Feltham (1993). The methyl red (MR)/Voges-Proskau- streptomycin (10 μg), sulphonamide (300 μg), tetracy- er (VP) reactions, indole test, citrate test, urease activ- cline (30 μg), tobramycin (10 μg) and vancomycin ity, amino acid decarboxylase (ADH, LDC, ODC), TDA (30 μg). Plates were incubated at 30°C for 24 h and the (tryptophan deaminase), ONPG (orthonitrophenyl-be- inhibition zone was interpreted. Ubiquinones (Q) were ta-D-galactopyranoside), assimilation of carbohydrates, extracted from freeze-dried cells with chloroform: metha- hydrolysis of esculin were determined with commer- nol (2：1) and purifi ed with a silica gel TLC (Merck cially product kit systems (API 20E, API 20NE and API No.1.05744). The purifi ed quinones were analyzed by 50CH, bioMérieux). Growth in 3, 4, 4.5 and 5% (w/v) HPLC (Komagata and Suzuki, 1987). Total cellular fatty NaCl, at different starting pH (3, 4, 5, 6, 7, 8, 9 and 10) acid analysis of cells grown on tryptic soy agar (TSA, and at different temperatures (4°C, 30°C, 37°C, 40°C Difco) for 48 h at 30°C was performed by Gas Liquid

Table 1. Differential characteristics of strain A3-3T and related Comamonas species.

Characteristics 1 2 3 4 5 6 7 b 8 c 9 d Cell shape Rd Rd Rd Rd Rd Rd Rd Rd Rd Flagella P P P P A P P P P Growth at 40°C++--w --++ Growth at pH 5 + - +++---- Urease + ----+ - ++ Nitrate reduction - +++++++- Tween 80 hydrolysis - ++++- ++- Citrate utilization + + + + --ww- Assimilation of : Arbutin ---+ ----- Glucose ---++--+ - Inositol ----+ ---- Mannitol ----+ ---- Tagatose ----+ ---- Caprate ---+ ----- Malate + + + + + - + - + Citrate ---w+- ++- Gluconate + - +++++-- Susceptibility to Ampicillin R R S R R S R R S Bacitracin R R S R S S R R S Cephalothin R S S R S S R R S Novobiocin S R S S R S S S R Penicillin G R R S R S S R R S Streptomycin S S S R R S R R S aG+C content (mol%) 69.6 61.0 64.0 62.5 66.0 64.0 61.6 61.4 68.4

Abbreviations: 1, A3-3T; 2, C. kerstersii LMG 3475T; 3, C. aquatica LMG 2370T; 4, C. testosteroni KCTC 2990T; 5, C. koreensis KCTC 12005T; 6, C. terrigena KCTC 2989T; 7, C. odontotermitis LMG 23579T; 8, C. thiooxidans DSM 17888T; 9, C. granuli KCTC 12199T. +, positive; w, weakly positive; -, negative; R, resistant; S, sensitive; Rd, rods; P, polar ﬂ agellum; A, absent; ND, no data. aData for the other type strains were obtained from Chang et al. (2002), De Vos et al. (1985), Tomaoka et al. (1987) and Young et al. (2008). bChou et al. (2007) and this study; cNarayan et al. (2010) and this study; dKim et al. (2008) and this study. 2012 Comamonas terrae sp. nov. 247

Table 2. Zone diameter (mm) a of antimicrobial disk susceptibility test for strain A3-3 and Comamonas species.

Strain LMG LMG KCTC KCTC KCTC Antibiotics A3-3T 3475T 2370T 12005T 2990T 2989T Ampicillin (10 μg) 0 0 34 ± 1.4 0 0 39 ± 1.4 Bacitracin (10 U) 0 0 13 ± 1.4 14 ± 1.4 0 16 ± 1.4 Carbenicillin (100 μg) 21 ± 1.4 26.5 ± 0.7 35 ± 1.4 33 ± 1.4 29.5 ± 0.7 35 ± 1.4 Cephalothin (30 μg) 0 29 ± 1.4 32.5 ± 0.7 13.5 ± 0.7 0 25 ± 1.4 Clindamycin (2 μg) 0 0 8.5 ± 0.7 0 0 0 Erythromycin (15 μg) 17.5 ± 0.7 21.5 ± 0.7 26.5 ± 0.7 23 ± 1.4 10.5 ± 0.7 13.5 ± 0.7 Gentamicin (10 μg) 18.5 ± 0.7 19.5 ± 0.7 19.5 ± 0.7 19 ± 1.4 8.5 ± 0.7 24 ± 1.4 Imipenem (10 μg) 34.5 ± 0.7 33 ± 1.4 32 ± 2.8 35 ± 1.4 32.5 ± 0.7 39 ± 1.4 Kanamycin (30 μg) 24.5 ± 0.7 20.5 ± 0.7 19.5 ± 0.7 24.5 ± 0.7 21 ± 1.4 21 ± 1.4 Novobiocin (5 μg) 7.5 ± 0.7 0 14.5 ± 0.7 0 11.5 ± 0.7 16.5 ± 0.7 Penicillin G (20 U) 0 0 32.5 ± 0.7 19 ± 1.4 0 39 ± 1.4 Streptomycin (10 μg) 10.5 ± 0.7 12.5 ± 0.7 10.5 ± 0.7 0 0 9.5 ± 0.7 Sulphonamide (300 μg) 31 ± 1.4 32.5 ± 0.7 35 ± 1.4 29 ± 1.4 30.5 ± 0.7 23 ± 1.4 Tetracycline (30 μg) 25.5 ± 0.7 31 ± 1.4 34 ± 1.4 33 ± 1.4 26 ± 1.4 31 ± 1.4 Tobramycin (10 μg) 19.5 ± 0.7 19 ± 1.4 18.5 ± 0.7 21 ± 1.4 11.5 ± 0.7 19 ± 1.4 Vancomycin (30 μg)000000

aValues are expressed as the means of two determinations.

Chromatography (GLC) according to the instructions Simmon citrate, urease, VP, hydrolysis of esculin, of the Microbial Identifi cation System (MIDI) Sherlock oxidation of glucose, assimilation of potassium glu- version 6.0 (Sasser, 1990) with the RTSBA6 MIDI data- conate, adipate and malate were positive. It grew at base. 40°C, in the presence of 4.5% (w/v) NaCl and at pH 5 DNA of strain A3-3T was extracted from 18‒24 h cells to 10 but it could not grow at 4°C and 50°C, in the pres- grown on TYEG agar and purifi ed by the method of ence of 5% (w/v) NaCl or at pH 4. Strain A3-3T was Saito and Miura (1963). DNA base composition was differentiated from the closest type strain, C. kerstersii determined by reversed-phase HPLC (Tamaoka and LMG 3475T by the growth at pH 5, urease, nitrate re- Komagata, 1984). The DNA-DNA hybridization was duction, Tween 80 hydrolysis, assimilation of potassi- conducted in multidilution well plates as reported by um gluconate and sensitivity to cephalothin and novo- Ezaki et al. (1989). The 16S rRNA gene was amplifi ed, biocin. This strain was also separated from the type purifi ed, and analyzed as described previously (Tana- species, C. terrigena KCTC 2989T(=LMG 1253T), and supawat et al., 2004). The derived sequence (1,487 bp) the other type strains, C. testosteroni KCTC 2990T was aligned with the selected sequences from the (=ATCC 11996 T), C. koreensis KCTC 12005T (=YH12T), GenBank/EMBL/DDBJ database employing CLUSTAL_X C. aquatica LMG 2370T, C. odontotermitis LMG 23579T, version 1.83 (Thompson et al., 1997). The alignment C. thiooxidans DSM 17888T and C. granuli KCTC was manually edited to remove gaps and ambiguous 12199T by their phenotypic characteristics and antibi- nucleotides prior to construction of a phylogenetic otic susceptibility as shown in Tables 1 and 2. Strain tree. The phylogenetic tree was constructed by using A3-3T, C. kerstersii LMG 3475T, C. aquatica LMG 2370T, the neighbor-joining method (Saitou and Nei, 1987) C. koreensis KCTC 12005T, C. testosteroni KCTC 2990T with the program MEGA 4 (Tamura et al., 2007). The and C. terrigena KCTC 2989T were sensitive to carben- confi dence value of branches of the phylogenetic tree icllin, erythromycin, gentamicin, imipenem, kanamy- was determined using bootstrap analyses (Felsen- cin, sulphonamide, tetracycline, and tobramycin but stein, 1985) based on 1,000 resamplings. resistant to vancomycin (Table 2). Additional charac- Strain A3-3T was an aerobic, Gram-negative rod with teristics are listed in the species description. a polar fl agellum. Colonies were circular and white, In the 16S rRNA gene sequence-based phylogenet- raised with entire margins, 1‒2.5 mm in diameter ic tree reconstructed according to the neighbor-joining and non pigmented. Catalase, oxidase, utilization of method, strain A3-3T was placed in a monophyletic 248 CHITPIROM et al. Vol. 58

Fig. 1. Neighbor-joining tree of 16S rRNA gene sequences showing the phylogenetic rela- tionships between strain A3-3T, Comamonas species and related taxa, Alcaligenes faecalis as the outgroup. Bootstrap values obtained with 1,000 bootstrap resamplings are shown on the branches.

Table 3. DNA-DNA relatedness of strain A3-3Tand related Comamonas species.

DNA-DNA relatedness (%) with labelled strainsa Strain A3-3T C. kerstersii LMG 3475T A3-3T 100.0 ± 0.45 12.3 ± 0.08 C. kerstersii LMG 3475T 9.2 ± 0.04 100.0 ± 0.19 C. aquatica LMG 2370T 6.0 ± 0.03 18.3 ± 0.06 C. testosteroni KCTC 2990T 30.6 ± 0.19 11.5 ± 0.08 C. koreensis KCTC 12005T 21.4 ± 0.09 12.8 ± 0.02 C. terrigena KCTC 2989T 19.6 ± 0.05 7.1 ± 0.02

aValues are expressed as the means of three determinations. cluster of the genus Comamonas (92.5‒96.9% similari- terrigena LMG 1253T (96.2%), C. zonglianii DSM ties) and was closed to related genera, Hydrogenopha- 22523T (96.0%), C. nitrativorans 23310T (95.7%), C. ga fl ava CCUG 1658T (93.4%), Delftia acidovorans denitrifi cans 123T (95.5%), C. granuli KCTC 12199T ACM 489T (94.4%), Brachymonas denitrifi cans AS-P1 T (95.1%), and C. badia IAM 14839T (92.5%). (95.0%), Giesbergeria sinuosa LMG 4393 T(95.9%) and The DNA-DNA relatedness was calculated by means Alcaligenes faecalis IAM 12369T (89.9%) (Fig.1). The of triplicate experiments. Strain A3-3T exhibited low strain was closely related to C. kerstersii LMG 3475T DNA-DNA relatedness (9.2‒30.6%) with C. kerstersii and C. aquatica LMG 2370T(96.9%), C. odontotermitis LMG 3475T, C. testosteroni KCTC 2990T, C. koreensis LMG 23579T(96.8%), C. testosteroni KCTC 2990T, C. KCTC 12005T, C. terrigena KCTC 2989Tand C. aquati- thiooxidans DSM 17888T and C. koreensis KCTC ca LMG 2370T (Table 3). C. kerstersii LMG 3475T ex- 12005T (96.5%), C. composti CC-YY287T (96.4%), C. hibited reciprocally low DNA-DNA relatedness (12.3%) 2012 Comamonas terrae sp. nov. 249

Table 4. Cellular fatty acid composition of strain A3-3T and related Comamonas species.

Fatty acids 1 23456 Saturated straight-chain C12:0 3.07 3.03 3.46 3.4 2.91 3.67 C14:0 tr 3.3 3.99 tr 0.86 3.25 C16:0 27.86 23.58 23.72 25.38 29.43 33.3 C17:0 0.6 ND 1.3 0.56 tr 1.0 C18:0 tr ND tr tr tr ND Unsaturated straight-chain C17:1 ω6c ND ND 0.59 ND ND ND C17:1 ω8c ND ND 0.59 ND tr tr Saturated branched-chain C17:0 CYCLO 16.72 1.35 ND 0.98 9.46 1.4 Unsaturated branched-chain C19:0 CYCLO ω8c 2.31 ND ND tr tr ND Hydroxylated fatty acids C10:0 3-OH 3.07 4.09 4.7 4.46 2.28 5.36 C14:0 2-OH tr ND ND 0.5 ND ND C15:0 2-OH tr ND ND tr ND ND C16:0 2-OH 4.67 ND ND 3.22 ND ND C16:1 2-OH 1.78 ND ND 1.25 ND ND Summed feature 3a 21.5 28.2 45.71 44.82 36.67 44.2 Summed feature 8b 16.36 34.09 14.73 15.58 16.39 13.12

Abbreviations: 1, A3-3T; 2, C. kerstersii LMG 3475T; 3, C. aquatica LMG 2370T; 4, C.testosteroni KCTC 2990T; 5, C. koreensis KCTC 12005T; 6, C. terrigena KCTC 2989T. a b Summed feature 3 contains C16:1 ω7c/C16:1 ω6c; Summed feature 8 contains C18:1 ω7c/

C18:1 ω6c; tr, trace (＜0.5%); ND, not detected. to strain A3-3T. The DNA-DNA relatedness of strain ness and cellular fatty acids as well as its phylogenetic A3-3T to its closest phylogenetic neighbors was well position, we conclude that the strain A3-3T represents below the 70% cut-off point recommended for the as- a novel species of the genus Comamonas, for which signment of the strains to the same genomic species the name Comamonas terrae sp. nov. is proposed. (Wayne et al., 1987). Based on the above DNA-DNA relatedness data, strain A3-3T was separated from the Description of Comamonas terrae sp. nov. related species of the genus Comamonas. Comamonas terrae sp. nov. (ter’rae. L. gen. n. terra Ubiquinone with eight isoprene unit (Q8, 93.4%) of the earth) was found to be the major quinone and Q-9 (4.9%) Aerobic, Gram-negative rods. Motile by a polar ﬂ a- and Q-7 (1.7%) were detected as minor quinones. Ma- gellum. Colonies were circular, formed white, raised with jor cellular fatty acids were C16:0 (27.86%) and C17:0 entire margins, 1‒2.5 mm diameter, without pigmenta- CYCLO (16.72%). This strain contained the same pro- tion. Non spore. Non-fermentative. Positive in tests for

ﬁ les of cellular fatty acids of C12:0, C16:0, C16:1 ω7c/ catalase, oxidase, utilization of citrate, urease, VP and C16:1 ω6c, C18:1 ω7c/C18:1 ω6c, and C10:0 3-OH as hydrolysis of esculin. Grows at 40°C in the presence of strains, C. kerstersii LMG 3475T, C. aquatica LMG 4.5% (w/v) NaCl and at pH 5 to11 but not at 4 or 50°C, 2370T, C. testosteroni KCTC 2990 T, C. koreensis KCTC in the presence of 5% (w/v) NaCl or at pH 4. Positive 12005T and C. terrigena KCTC 2989T but showed the assimilation for potassium gluconate, adipate and different amount fatty acids of C17:0 CYCLO, C19:0 CY- malate. Negative in tests for MR, amino acid decar- CLO ω8c, C16:0 2-OH and C16:1 2-OH from the related boxylase (ADH, LDC, ODC), ONPG, hydrolysis of TDA, Comamonas species (Table 4). lysine, arginine, gelatin, starch, Tween 80; indole, ni-

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